药学学报, 2022, 57(5): 1459-1464
引用本文:
刘春雨, 于传飞, 李欣, 付志浩, 崔永霏, 郭璐韵, 王兰. 利用实验设计优化和建立抗CD38单克隆抗体ADCP生物学活性测定方法[J]. 药学学报, 2022, 57(5): 1459-1464.
LIU Chun-yu, YU Chuan-fei, LI Xin, FU Zhi-hao, CUI Yong-fei, GUO Lu-yun, WANG Lan. A method for measuring the ADCP potency of anti-CD38 monoclonal antibody[J]. Acta Pharmaceutica Sinica, 2022, 57(5): 1459-1464.

利用实验设计优化和建立抗CD38单克隆抗体ADCP生物学活性测定方法
刘春雨1#, 于传飞1#, 李欣2, 付志浩1, 崔永霏1, 郭璐韵1, 王兰1*
1. 中国食品药品检定研究院单克隆抗体产品室, 国家卫生健康委员会生物技术产品检定方法及其标准化重点实验室/国家药品监督管理局生物制品质量研究与评价重点实验室, 北京 102629;
2. 广州市药品检验所, 广州 510160
摘要:
利用实验设计(design of experiment,DoE)方法建立抗CD38单抗(monoclonal antibody,mAb)的抗体依赖性细胞吞噬作用(antibody-dependent cell-mediated phagocytosis,ADCP)生物学活性检测方法。以Jurkat/NFAT/CD32a-FcεRIγ转基因细胞系作为效应细胞,Daudi细胞系作为靶细胞,通过荧光素酶检测系统(BrightGloTM Luciferase Assay system)检测抗CD38单抗的ADCP生物学活性,并运用DoE方法进行实验参数优化。结果显示,抗CD38单抗在该方法中存在量效关系,且符合四参数方程:y =(A-D)/[1+(x/CB]+D,方法经统计学实验设计对多个实验参数进行筛选和优化,确定抗体工作浓度为800~20.81 ng·mL-1,靶细胞和效应细胞的接种量分别为每孔7.5×104和2.5×104个,诱导时间为6 h。该方法具有良好的专属性;5个不同组回收率样本经3次检测,相对效价分别为(59.97 ±4.74)%、(82.44 ±5.15)%、(110.69 ±11.71)%、(129.23 ±5.22)%和(162.15 ±3.66)%;回收率在103%~120%,RSD均小于11%,线性检测范围为50%~150%。本研究利用DoE设计成功筛选、优化和建立基于报告基因的抗CD38单抗ADCP生物学活性测定方法,该方法具有良好的专属性、重复性和准确性,可作为抗CD38单抗ADCP生物学活性的评价方法。
关键词:    抗CD38单抗      抗体依赖性细胞吞噬作用      生物学活性      实验设计      Plackett-Burman设计      优化      验证     
A method for measuring the ADCP potency of anti-CD38 monoclonal antibody
LIU Chun-yu1#, YU Chuan-fei1#, LI Xin2, FU Zhi-hao1, CUI Yong-fei1, GUO Lu-yun1, WANG Lan1*
1. Division of Monoclonal Antibody, National Institutes for Food and Drug Control, NHC Key Laboratory of Research on Quality and Standardization of Biotech Products, NMPA Key Laboratory for Quality Research and Evaluation of Biological Products, Beijing 102629, China;
2. Guangzhou Insititue for Drug Control, Guangzhou 510160, China
Abstract:
A method to measure the antibody-dependent cell-mediated phagocytosis (ADCP) potency of anti-CD38 mAb was developed based on design of experiment (DoE) with a Jurkat/NFAT/CD32a-FcεRIγ transgenic cell line as the effector cell, the Daudi cell line as the target cells, and luciferase as the detection system. The DoE method was used for optimization of experimental parameters and methodological validation. The results show that anti-CD38 mAb exhibits a dose-response relationship with the following four-parameter equation:y=(A-D)/[1 + (x/C)B]+ D. Several experimental parameters were optimized by statistical experimental design and determined as follows:the working concentration of anti-CD38 mAb was 800-20.81 ng·mL-1, the density of the target cells was 7.5×104 per well, and the density of effector cells was 2.5×104 per well, with an induction time of 6 h. The method showed good specificity. The recovery rate for samples from 5 different groups showed that the relative potencies of anti-CD38 mAb were (59.97 ±4.74)%, (82.44 ±5.15)%, (110.69 ±11.71)%, (129.23 ±5.22)% and (162.15 ±3.66)%. The recoveries ranged from 103% to 120% and the RSDs of the above results were all less than 11%. The linear detection range was 50%-150%. Based on DoE design, this method for measuring ADCP potency of anti-CD38 mAb was optimized and validated with good specificity, repeatability and accuracy. This method can be used for evaluation of ADCP biological activity of anti-CD38 mAbs.
Key words:    anti-CD38 monoclonal antibody    antibody-dependent cell-mediated phagocytosis    biological activity    design of experiment    Plackett-Burman design    optimization    validation   
收稿日期: 2021-11-10
DOI: 10.16438/j.0513-4870.2021-1613
基金项目: “重大新药创制”国家科技重大专项(2018ZX09736-016-007);中山市重大科技专项(210204163866513).
通讯作者: 王兰,E-mail:wanglan@nifdc.org.cn
Email: wanglan@nifdc.org.cn
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